The Aurora kinase Ipl1 is necessary for spindle polebody cohesion during budding yeast meiosis

Katelan Shirk1, Hui Jin1, Thomas H. Giddings, Jr2, Mark Winey2 and Hong-Guo Yu1,*1Department of Biological Science, The Florida State University, Tallahassee, FL 32306-4370, USA2Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, CO 80309, USA*Author for correspondence (hyu@bio.fsu.edu)

Accepted 3 May 2011Journal of Cell Science 124, 2891–2896� 2011. Published by The Company of Biologists Ltddoi: 10.1242/jcs.086652

SummaryIn budding yeast, the microtubule-organizing center is called the spindle pole body (SPB) and shares structural components with the

centriole, the central core of the animal centrosome. During meiotic interphase I, the SPB is duplicated when DNA replication takesplace. Duplicated SPBs are linked and then separate to form a bipolar spindle required for homolog separation in meiosis I. Duringinterphase II, SPBs are duplicated again, in the absence of DNA replication, to form four SPBs that establish two spindles for sister-

chromatid separation in meiosis II. Here, we report that the Aurora kinase Ipl1, which is necessary for sister-chromatid cohesion, is alsorequired for maintenance of a tight association between duplicated SPBs during meiosis, which we term SPB cohesion. Premature lossof cohesion leads to SPB overduplication and the formation of multipolar spindles. By contrast, the Polo-like kinase Cdc5 is necessaryfor SPB duplication and interacts antagonistically with Ipl1 at the meiotic SPB to ensure proper SPB separation. Our data suggest that

Ipl1 coordinates SPB dynamics with the two chromosome segregation cycles during yeast meiosis.

Key words: Spindle pole body (SPB), Centrosome, SPB duplication, Aurora kinase, Ipl1, Polo-like kinase, Meiosis

IntroductionStructurally and conceptually, the budding yeast spindle pole

body (SPB) is best known from vegetative cells (Adams and

Kilmartin, 2000; Jaspersen and Winey, 2004). It is embedded in

the nuclear envelope as a layered structure (Moens and Rapport,

1971; Byers and Goetsch, 1974). Duplication of the SPB is

initiated with the deposition of the satellite at the distal end of the

half bridge, a specialized membrane structure attached to the SPB

central plaque (Byers and Goetsch, 1974). One of the major

components of the satellite is the SPB core component, Spc42

(Donaldson and Kilmartin, 1996; Bullitt et al., 1997). Duplicated

SPBs form a side-by-side configuration and are tethered together

by the bridge, which is severed or disassembled upon the

formation of a bipolar spindle, promoted by the actions of the

mitotic cyclin Cdk and the Polo-like kinase Cdc5 (Kilmartin,

2003; Jaspersen et al., 2004; Crasta et al., 2008). Two known

structural components of the half bridge are Sfi1 and Cdc31,

homologs of which are essential components in the vertebrate

centriole (Li et al., 2006). The meiotic SPB resembles its mitotic

counterpart (Moens and Rapport, 1971; Straight et al., 2000) and

is perhaps regulated in a similar fashion, but how it is

reduplicated at interphase II to coordinate meiotic chromosome

segregation remains unknown.

In budding yeast, the Aurora kinase Ipl1 is required for the

protection of sister-chromatid cohesion during meiosis (Monje-

Casas et al., 2007; Yu and Koshland, 2007). Ipl1 is the founding

member of the Aurora kinase family and is closely related to the

Aurora B kinase found in higher eukaryotes (Chan and Botstein,

1993). Here, we report that Ipl1 is also required for maintenance

of a tight association between duplicated SPBs and prevents SPB

overduplication at interphase II, revealing that Ipl1 plays a role

similar to that of the Aurora A kinase, being important for

centrosome propagation. As in animal cells, where centriole

duplication depends on Plk1 (Tsou et al., 2009), licensing of SPB

duplication requires Cdc5 in budding yeast meiosis.

Results and DiscussionIpl1 is required for SPB cohesion during meiosis

To investigate the dynamic separation of duplicated SPBs in

meiosis I (MI), we developed a live-cell culture method of

inducing yeast meiosis on a glass slide and observed Spc42–GFP-

marked SPBs by fluorescence microscopy (Fig. 1A). In wild-type

cells, SPBs were tightly associated after duplication, primarily

forming a single Spc42–GFP focus (Fig. 1A). Immediately

before their separation, SPBs were resolved by light

microscopy as two distinguishable foci (Fig. 1A, t50), which

we refer to as a doublet SPB configuration. For live-cell

microscopy, we defined time zero as the point immediately

before SPB separation in MI (,4 hours after induction of

meiosis), so that we could objectively examine and compare SPB

separation and meiotic progression. In contrast to those from the

wild type, SPBs in Ipl1-depleted (PCLB2-IPL1) cells were mostly

associated at a greater distance and formed the doublet

configuration well before their complete separation (Fig. 1B,C),

suggesting that the sister SPBs are less cohesive in the absence of

Ipl1. To examine the meiotic SPB at a higher resolution, we

examined serially sectioned cells by electron microscopy

(Fig. 1D–F). Consistent with our light-microscopy observations,

duplicated SPBs were linked by the bridge and were tightly

associated for an extended period of time in MI (Fig. 1), a

situation we termed SPB cohesion. By contrast, sister SPBs were

positioned significantly further apart from each other with a

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collapsed bridge before their complete separation in Ipl1-depleted cells (Fig. 1D, 118 nm in the wild type, 162 nm in themutant). In addition, the SPB-associated nuclear envelope oftenbecame invaginated (Fig. 1F,F2–F6). Even in these cells, the

layered structure of SPBs resembled those of the wild type(Fig. 1F). The average widths of SPB layers from wild-type andIpl1-depleted cells did not differ significantly during MI, but

mutant cells showed a greater variation in SPB size (Fig. 1E).Together, these results show that the layered SPB structureappears normal in Ipl1-depleted meiotic cells, but sister SPBs are

positioned further apart, suggesting that Ipl1 is required for themaintenance of bridge integrity and therefore SPB cohesion.

Ipl1 is required for accurate SPB duplication at interphase II

After SPBs separate in MI, they undergo a second round ofduplication at interphase II. In wild-type cells, four SPBs were

formed after completion of meiosis II (MII) (Fig. 2A,B). Bycontrast, more than 30% of Ipl1-depleted meiotic cells had

formed five or more SPBs 6 hours after induction, a time that

corresponded to MII (Fig. 2A,B). These supernumerary SPBs

contained the meiotic plaque component Mpc54 (supplementary

material Fig. S1), which is characteristic of MII SPBs (Knop andStrasser, 2000). In addition, inactivation of the Ipl1 kinase

activity in the ipl1-as5 allele (Pinsky et al., 2006) during meiosis

also led to the formation of extra Spc42 foci (Fig. 2C). Together,

these results suggest that sister SPBs prematurely lose cohesion

and subsequently overduplicate in the absence of Ipl1 activityduring meiosis.

To determine when Ipl1 is required for SPB duplication andwhether SPBs become fragmented in Ipl1-depleted cells, we

arrested the cells at prophase I by eliminating the production of

Ndt80 by means of the GAL-NDT80 allele (Carlile and Amon,

Fig. 1. Requirement for Ipl1 for SPB cohesion during yeast

meiosis. (A,B) Fluorescence live-cell microscopy images

showing the morphology of duplicated sister SPBs in wild-type

(WT, HY1423C) and PCLB2-IPL1 (HY1423) cells in MI. SPBs are

marked by Spc42–GFP. Time zero was defined as the point

immediately before sister-SPB separation in MI. The time in

minutes is shown above each frame. Projected images are shown.

Three-dimensional histograms show Spc42–GFP intensity. Pixel

intensity counts are shown to the right. Scale bar: 0.5 mm.

(C) Quantification of SPB doublet formation. Cells were induced

to undergo synchronous meiosis, fixed at the indicated times and

visualized under a fluorescence microscope. At least 100 cells

were counted for each time point. (D,E) SPB-to-SPB distance and

SPB plaque width in MI. Cells were induced to undergo

synchronous meiosis, subjected to high-pressure freezing and

freeze substitution, serially sectioned and visualized with electron

microscopy. MI cells with side-by-side SPBs were identified;

SPB-to-SPB distance and plaque width were determined from

single sections. WT, n59; mutant, n57. Error bars show s.d.

(F) Representative images showing sister-SPB configuration from

WT (F1) and mutant (F2–F6) cells. Five serial sections are shown

for the mutant. Note that the SPB-associated nuclear envelope

became invaginated in the mutant. NE, nuclear envelope.

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2008). We then observed SPB separation with and without the

Ipl1 kinase activity (Fig. 2D). Sister SPBs failed to separate in

cells arrested at prophase I, but inactivation of Ipl1 led to SPB

separation in ,50% of these cells, supporting the idea that Ipl1 is

required for maintaining SPB cohesion. Importantly, these

separated SPBs did not commit to duplication unless Ndt80

was reintroduced and cells proceeded through meiosis (Fig. 2D).

These data suggest that supernumerary SPB formation in Ipl1-

depleted cells is less likely to be due to SPB fragmentation. We

therefore conclude that the Aurora kinase Ipl1 is required for SPB

cohesion and prevents SPB overduplication at interphase II.

Ipl1 prevents multipolar spindle formation

To determine whether overduplicated SPBs in ipl1 mutant cells

function to nucleate microtubules, we observed SPB dynamics and

spindle formation (Fig. 3A,B). In wild-type cells, sister SPBs

remained tightly associated after duplication (Fig. 3A). SPBs were

separated to form a 2- to 3-mm-long spindle during metaphase I

(Fig. 3A,C). Approximately 30 minutes after SPB separation, the

spindle elongated to reach a length of ,6 mm at anaphase I. Then

the pole-to-pole distance decreased slightly during an ,8-minute

window, before SPBs separated again in MII (Fig. 3C). On

average, the interval from the beginning of MI SPB separation to

that of MII was 40 (¡ 3) minutes (n514). Reduplicated SPBs were

kept together before the formation of the MII spindle (Fig. 3A,

from t540 minutes to t546 minutes), as sister SPBs were in MI. In

Ipl1-depleted meiotic cells, well before MI separation, sister SPBs

formed the doublet configuration (Fig. 3B, t5–24 minutes).

Separated SPBs formed a bipolar spindle. Notably, mutant cells

lacked a clear spindle-elongation phase (anaphase I); instead, 30

(¡8) minutes (n512) after MI SPB separation, these cells started

to form a third Spc42–GFP focus, apparently originating from an

existing SPB; then additional Spc42–GFP foci formed (Fig. 3B,C).

The newly formed SPBs were initially present at a very low Spc42–

GFP fluorescence intensity (Fig. 3B, t539 min); they grew to an

intensity similar to that of the old MI SPBs in ,20–30 minutes

(Fig. 3B). All Spc42–GFP foci formed in Ipl1-depleted cells were

able to nucleate microtubules, often resulting in multipolar spindles

(Fig. 3B), demonstrating that these SPBs are functional in

microtubule organization. The SPB morphology appears normal

in Ipl1-depleted cells (Fig. 1; supplementary material Fig. S2),

suggesting that these SPBs are fully formed. Collectively, our data

support the idea that Ipl1 is required for faithful duplication of

existing SPBs and prevention of multipolar spindle formation.

Cdc5 licenses SPB duplication

We hypothesized that the completion of SPB separation depends

on the formation of a bipolar spindle between the SPBs. The Polo

kinase, Cdc5 in yeast, is implicated in SPB separation in

vegetative yeast cells and centriole separation in animal cells

(Crasta et al., 2008; Tsou et al., 2009). Cdc5, C-terminally tagged

with GFP, appeared in the nucleus ,20 minutes before MI SPB

separation in wild-type cells, forming foci that were not

associated with SPBs (Fig. 4A, from t5–18 to t5–4 minutes,

arrows). Cdc5 was concentrated on the SPB during MI SPB

separation (Fig. 4A, t50 minutes, arrows). By contrast, in Ipl1-

depleted cells, the complete separation of sister SPBs in MI

started at the appearance of Cdc5–GFP, as detected by

fluorescence microscopy (Fig. 4B,C), indicating that, upon

Cdc5 production, cells immediately initiated SPB separation

and bipolar spindle formation. In the mutant cell shown, five

Tub4 foci formed 66 minutes after SPB separation and all were

enriched with Cdc5–GFP (Fig. 4B). Because the production of

Cdc5 appeared at the usual time in Ipl1-depleted cells (Fig. 4D),

our data suggest that SPB cohesion prevents premature SPB

separation and that Cdc5 is a regulator of this process.

Fig. 2. Requirement for Ipl1 for SPB reduplication during yeast meiosis.

(A) Fluorescence live-cell microscopy showing SPB segregation in WT

(HY1675) and PCLB2-IPL1 (HY1886) cells during yeast meiosis. SPBs are

marked by Spc42–GFP and Tub4–mApple. *Duplicated sister-SPBs formed a

doublet configuration in PCLB2-IPL1 cells before separation in MI. Scale bar:

2 mm. (B) The number of Spc42 foci in fixed samples from WT (left) and

PCLB2-IPL1 (right) cells. One and two SPB cells were grouped as MI; three

and four were grouped as meiosis II (MII); five or more represents cells with

overduplicated SPBs. Averages from two independent experiments are

shown. (C) Spc42–GFP focus formation in the ipl1-as5 mutant (HY2486)

during meiosis. Addition of 1-NM-PP1 inhibits the Ipl1 kinase activity.

DMSO treatment (left) serves as a control. Figure symbols are as in B.

(D) The execution point of Ipl1 on SPB cohesion and duplication. Yeast cells

(HY2627) were induced to undergo synchronous meiosis, subjected to four

different treatments and fixed at the indicated times for fluorescence

microscopy. Addition of b-estradiol induced the production of Ndt80. Arrows

indicate the time of addition.

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Fig. 3. Microtubule spindle formation during

meiosis. (A,B) Fluorescence live-cell microscopy

showing SPBs and microtubule spindle dynamics in

WT (HY1737) and PCLB2-IPL1 (HY1738) cells

during yeast meiosis. SPBs are marked by Spc42–

GFP and microtubules by Tub1–mApple. Time zero

was defined as the point of SPB separation in MI.

The time in minutes is shown below each frame.

The time-lapse was 2 minutes for the wild-type cell

and 3 minutes for the PCLB2-IPL1 cell. Movies are

provided as supplemental data (supplementary

material Movies 1 and 2). Note that sister SPBs

form a doublet configuration before MI separation

in PCLB2-IPL1 cells. Red, Spc42; green, Tub1.

Scale bar: 2 mm. (C) Spindle length as determined

by pole-to-pole distance in WT and PCLB2-IPL1

cells, as shown in A and B. The distance in three

dimensions was measured. MI spindle, purple; MII

spindle, other colors.

Fig. 4. Promotion of SPB duplication by

Cdc5 at interphase II. (A,B) Live-cell

fluorescence microscopy showing Cdc5

localization in WT (HY1993) and PCLB2-IPL1

(HY2037) cells during meiosis. Cdc5 was

tagged with GFP. SPBs are marked by

Tub4–mApple. Projected images are

shown. Arrows point towards SPB and

Cdc5 foci. Red, Tub4; green, Cdc5. Scale

bar: 2 mm. (C) Relative intensity of Cdc5–

GFP in live meiotic cells. The Cdc5–GFP

intensity of an area made up of 400 pixels is

plotted against time. This area is centered

on the SPBs. The background intensity of

Cdc5 is about 110. (D) Western blot

showing Cdc5–GFP production during

yeast meiosis from WT (HY1993),

PCLB2-IPL1 (HY2037) and PCUP1-CDC5

(HY2076) cells. The level of Tub2 serves

as a loading control. (E) The number of

Tub4–GFP foci in fixed samples from WT

(HY1881C), PCLB2-CDC5 (HY2161),

PCLB2-CDC5 PCLB2-IPL1 (HY2458) and

PCUP1-CDC5 (HY2169) cells. Averages

from two independent experiments

are shown.

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To determine whether Cdc5 is required for SPB duplication at

interphase II, we depleted Cdc5 by means of the PCLB2-CDC5

allele (Clyne et al., 2003; Lee and Amon, 2003). In Cdc5-

depleted cells, SPBs separated in MI but failed to duplicate at

interphase II (Fig. 4E). By contrast, overproduction of Cdc5 by

the PCUP1-CDC5 allele dramatically increased the number of

meiotic cells with supernumerary SPBs (Fig. 4D,E). In cells

depleted of both Ipl1 and Cdc5, ,40% MI SPBs were duplicated

to form three or four but not five SPBs (Fig. 4E), suggesting that

other factors, in addition to Cdc5, can also promote SPB

duplication. Together, these data support the idea that Ipl1

protects SPB cohesion and prevents SPB overduplication,

whereas Cdc5 promotes SPB duplication at interphase II.

We propose that SPB cohesion restricts SPB duplication at

interphase II, analogous to the notion that engaged centrioles are not

licensed for duplication in vertebrates (Tsou and Stearns, 2006).

Precocious separation of sister SPBs, presumably due to the collapse

of the bridge, licenses them to undergo additional rounds of

duplication at interphase II when SPBs are competent for

duplication. Meiotic SPB duplication requires the antagonistic

interaction between two important cell-cycle regulators, Ipl1 and

Cdc5, which have been implicated in numerous activities involving

chromosomes and the spindle microtubules through phosphorylation

of a variety of cellular substrates. In yeasts, Ipl1 is the only Aurora

kinase that functions both on the spindle and at the spindle poles,

whereas in metazoans Aurora kinase functions are differentiated,

with Aurora A the kinase predominately located at the poles (Barr

and Gergely, 2007; Lukasiewicz and Lingle, 2009). In this regard,

the kinase activity of Ipl1 in protecting SPB cohesion and preventing

SPB overduplication might resemble that of Aurora A kinase in

higher eukaryotes. Meanwhile, the Polo-like kinase Cdc5 is required

for licensing SPB duplication, presumably by promoting the

dissolution of SPB cohesion, as it does with the vertebrate

centriole (Tsou et al., 2009). Conceivably, Ipl1 and Cdc5 regulate

bridge integrity and SPB cohesion by controlling the

phosphorylation status of their substrates at the SPB during

budding yeast meiosis.

Materials and MethodsYeast strains and culture methods

Yeast strains used in the study are diploid SK1 derivatives (supplementary material

Table S1). The following mutant alleles have been previously described: PCLB2-

IPL1 (Yu and Koshland, 2007), PCLB2-CDC5 (Lee and Amon, 2003), ipl1-as5

(Pinsky et al., 2006), and GAL4.ER and GAL-NDT80 (Carlile and Amon, 2008).

We used a PCR-based gene-replacement method to construct PCUP1-CDC5 byreplacing the endogenous CDC5 promoter with the CUP1 promoter (Jin et al.,

2009). A similar PCR-based method was used to construct SPC42-GFP, CDC5-

GFP, MPC54-GFP, TUB1-mApple, and TUB4-mApple. PCR primer information isavailable upon request.

Yeast cells were grown at 30 C̊ with standard culture methods. Before yeastcells were induced to enter meiosis in 2% potassium acetate, they were grown in

the YPA medium with vigorous shaking for about 12 hour, to an optical density

(l5600 nm) of 1.5. To induce PCUP1-CDC5 expression during meiosis, we added60 mM (final concentration) of CuSO4 to the sporulation medium after induction of

meiosis. Inactivation of ipl1-as5 (Pinsky et al., 2006) was induced by addition of

100 mM (final concentration) of 1-NM-PP1 to the sporulation medium afterinduction of meiosis. To induce GAL-NDT80 expression, we added 100 mM b-

estradiol (final concentration) 6 hours after induction of meiosis (Fig. 2D). For

monitoring of SPB formation, aliquots of cells were withdrawn at the indicatedtimes, fixed with 1% formaldehyde for 1 hour at room temperature, washed twice

with 16 PBS and visualized under a fluorescence microscope.

For live-cell microscopy, we used a concave glass slide as a culture chamber,

which was filled with 2% agarose dissolved in 2% potassium acetate. The agarose

pad was solidified for 5 minutes at room temperature before use. About 1.5 mlyeast culture was laid on top of the agarose pad then sealed with a glass coverslip.

The slide was temperature balanced for 15 minutes at 30 C̊ before microscopy.

Fluorescence microscopy

Live-cell microscopy was carried out on a DeltaVision imaging system (AppliedPrecision). All live-cell images were acquired at 30 C̊ with a 606 (NA51.41)objective lens. Seven or eight z-stacks were collected at each time point. Each

optical section was 1 mm thick. The exposure time for each optical section was setbetween 60 and 100 ms. Fixed cells were visualized under an epifluorescencemicroscope (AxioImager, Zeiss) with a 1006 objective lens (NA51.40).

Electron microscopy

Yeast aliquots (5 ml) were collected 4 and 6 hours after induction of meiosis.Blocks of cells were prepared by a high-pressure freezing and freeze-substitutionmethod, as described previously (Winey et al., 2005). Serial sections of embeddedcells were obtained and visualized under a transmission electron microscope(Philips, CM10).

Data analysis and image display

Raw data collected from the DeltaVision imaging system were deconvolved withSoftWorx (Applied Precision). The three-dimensional pole-to-pole length of theSPB (Fig. 3) was determined with the measurement tool provided by SoftWorx.Optical sections were projected into two dimensions for display. Projected imageswere used to generate the histograms shown in Fig. 1A and Fig. 4B. An areacomposed of 400 pixels centered on the SPBs is shown, and the pixel size is0.1070 mm.

Western blot

Western blotting was performed as previously described (Jin et al., 2009). Cdc5–GFP was detected by an anti-GFP antibody (Cat#632569, Clontech). The level ofTub2 (b-tubulin) served as a loading control.

We thank A. Amon, S. Biggins and M. Davidson for providingyeast strains and reagents. M. Avey and C. Clarissa providedtechnical assistance. A. B. Thistle assisted with text editing. Thiswork was supported in part by NIH R01GM51312 to M.W. and bythe Florida Biomedical Research Program (08BN-08) and theNational Science Foundation (MCB-0718384) to H.Y. Deposited inPMC for release after 12 months.

Supplementary material available online at

http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.086652/-/DC1

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